Tuberculosis is a devastating disease affecting 100 million individuals around the world, and the causative agent Mycobacterium Tuberculosis (Mtb) has become increasingly resistant to the available antibiotics. Consequently, it is vital to identify Mtb enzymes that are essential for its viability and to find drugs that selectively inhibit their functions. In collaborative studies, we have established by genetic approaches that clpP1 and clpP2 genes, which appear to encode intracellular proteases, are essential for the viability and infectivity of Mtb. Our laboratory has recently shown that Mtb ClpP1 and ClpP2 proteins are components of a single proteolytic complex, and we have developed methods to express, isolate and assay the active enzyme, which has a number of novel structural and regulatory properties. In E. Coli, the homologous enzyme, ClpP, is a well- characterized protease complex that is composed of 14 identical subunits and functions in protein degradation as a part of the ATP-dependent proteolytic complexes. Mtb ClpP1P2, however, is a 14-mer composed of a ClpP1 and a ClpP2 heptameric ring. In addition, we identified several small molecule (peptides or peptide derivatives) activators that are required for the formation of the active mixed ClpP1P2 complex and for its activity (even in the absence of a regulatory ATPase). We identified a number of fluorescent peptide substrates that can be used in a sensitive, reliable assay and showed that active sites on the ClpP1 and ClpP2 rings differ in substrate preference. Since ClpP is not present in the cytoplasm of mammalian cells, where protein breakdown involves very different enzymes (by the ubiquitin-proteasome pathway or in lysosomes), specific inhibitors of ClpP or of its novel activation mechanism are highly attractive drug targets. Our major goals will be to further characterize Mtb ClpP1P2 and its properties and to adapt our enzymatic assay to a high throughput format that will allow us to screen for small molecule inhibitors and activators. Because a class of antibiotics has been reported that causes uncontrolled activation of the ClpP protease in B.subtilis with toxic consequences, we shall also screen for agents that activate ClpP. These inhibitors or activators could then serve as lead compounds in a drug development program. PUBLIC HEALTH RELEVANCE: Tuberculosis is a devastating disease affecting 100 million individuals around the world. This bacterium has become increasingly resistant to the available antibiotics. Our collaborator Dr. Eric Rubin and co-workers have found that genes (clpP1 and clpP2) encoding protease ClpP are essential for the viability and infectivity of Mycobacterium Tuberculosis (Mtb). Therefore this enzyme is a highly attractive drug target, especially since it does not exist in the cytosol of mammalian cells. Our laboratory succeeded in isolating and characterizing this member of ClpP family, which has many novel biochemical properties. Our major goals will be to optimize a high throughput assay of this enzyme that will allow screen for small molecule inhibitors (or activators) that might serve as lead compounds in a drug development program.